Low interleukin-10 level indicates a good prognosis in Salmonella enterica serovar typhimurium-induced pediatric hemophagocytic lymphohistiocytosis:A case report

BACKGROUND Secondary hemophagocytic lymphohistiocytosis(sHLH)triggered by Salmonella enterica serovar Typhimurium is rare in pediatric patients.There is no consensus on how to treat S.typhimurium-triggered sHLH.CASE SUMMARY A 9-year-old boy with intermittent fever for 3 d presented to our hospital with positive results for S.typhimurium,human rhinovirus,and Mycoplasma pneumoniae infections.At the time of admission to our institution,the patient’s T helper 1/T helper 2 cytokine levels were 326 pg/mL for interleukin 6(IL-6),9.1 pg/mL for IL-10,and 246.7 pg/mL for interferon-gamma(IFN-γ),for which the ratio of IL-10 to IFN-γwas 0.04.In this study,the patient received meropenem,linezolid,and cefoperazone/sulbactam in combination with high-dose methylprednisolone therapy(10 mg/kg/d for 3 d)and antishock supportive treatment twice.After careful evaluation,this patient did not receive HLH chemotherapy and recovered well.CONCLUSION S.Typhimurium infection-triggered sHLH patient had a ratio of IL-10 to IFN-γ≤1.33,an IL-10 concentration≤10.0 pg/mL,and/or an IFN-γconcentration≤225 pg/mL at admission.Early antimicrobial and supportive treatment was sufficient,and the HLH-94/2004 protocol was not necessary under these conditions.

Deletion of Salmonella enterica serovar typhimurium sipC gene

Objective:To construct a novel plasmid as Salmonella enterica serovar typhimurium(S.typhimurium)sip C gene knockouts candidate.Methods:In this research,50upstream and 30downstream regions of S.typhimurium sip C gene and kanamycin gene were PCR amplified.Each of these DNA fragment was cloned into p GEM T-easy vector.The construct was confirmed by PCR and restriction digest.Results:PCR amplified 320,206 and 835 bp DNA fragments were subcloned into p ET-32 vector resulting with a plasmid called p ET-32-sip C up-kan-sip C down.Conclusions:The new plasmid(p ET-32-sip C up-kan-sip C down)is useful for genetic engineering and for future manipulation of S.typhimurium sip C gene.

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL^(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.

Enhancing chloramphenicol and trimethoprim in vitro activity by Ocimum sanctum Linn.(Lamiaceae) leaf extract against Salmonella enterica serovar Typhi

Objective:To evaluate the antibacterial activity of Ocimum sanctum(O.sanctum) leaf extract, alone,and in combination with chloramphenicol(C) and trimethoprim(Tm) against Salmonella enterica serovar Typhi(S.typhi).Methods:The antibacterial activity of ethanolic extract of tulsi, 0.sanctum,leaf(TLE:500μg) for 23 S.typhi isolates was determined following agar diffusion. The C(30μg) and Tm(5μg) activity alone and in combination with TLE(250μg) was determined by disk diffusion.The zone diameter of inhibition(ZDI) for the agents was recorded, and growth inhibitory indices(Glls) were calculated.Results:The S.typhi isolates(n=23),which were resistant to both C(ZDI 6 mm) and Tm(ZDI 6 mm),had TLE(500μg) ZDIs 16-24 mm.The ZDIs of C and Tm were increased up to 15-21 mm and 17-23 mm,respectively,when TLE(250μg) was added to the C and Tm discs.The Glls ranged 0.789-1.235 and 0.894-1.352,due to combined activity against S.typhi isolates,of C and TLE and Tm and TLE.respeclivelv.Conclusions:The data suggest that TLE,in combination with C and Tm,had synergistic activity for S.typhi isolates, and hence O.sanclum is potential in combating S.typhi drug resistance,as well promising in the development of non-antibiotic drug for S.typhi infection.

Identification of a toxin coding fragment in pBSSB1, a linear plasmid from Salmonella enterica serovar Typhi that can stabilize a multicopy plasmid

Objective: To identify the region conferring stability to pBSSB2(a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame(ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB(plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.

Approach to Epidemiological Mechanism of Infection or Colonization of Egg-Laying Chicken Farms by <i>Salmonella enterica</i>Serovar Enteritidis (SE) Becoming the Main Source of Contamination in Food Poisoning (Review)

Salmonella enterica serovar Enteritidis (SE)-induced diarrhea in humans is the typical non-typhoid diarrhea. It develops acutely or subacutely and may be fatal. This SE infectious disease suddenly became a major public health issue worldwide in the 1980s. The main causative food material of SE food poisoning is chicken eggs, and many outbreaks of food poisoning caused by chicken eggs occurred throughout the world. SE epidemics occurred in layer farms, and this was the main cause of SE-induced food poisoning in humans. The major subject of our epidemiological study described in this report is why SE-contaminated eggs became the main causative food. In this study, we focused on difference of molecular expression for farm-isolated SEs. That is because recent studies have demonstrated that O-antigen enlargement may be related to pathogenicity in mice as well as 22-kDa polypeptide-expression (SEp22). We have discovered that many SE strains isolated from chicken farms do not express SEp22, and a deficiency or decreased level of cellular antigen 0-12 in SE strains isolated from chicken farms was clarified in a report. Additionally, SEp22 was deficient in SE strains passaged through chickens, whereas SEp22 was expressed at a high level in SE strains passaged through mice. These findings suggest that SE infection and retention more effectively occur in layer farms than in other animal maintenance environments, which may be a basis of the epidemiological hypothesis to explain the high-levelproduction of SE-contaminated eggs (the presence of mice may be the basis of the retention of SE infection in layer farms, and this may also be the mechanism causing the high-level production of SE-contaminated eggs).

Response of Salmonella enterica serovar Typhimurium to alginate oligosaccharides fermented with fecal inoculum:integrated transcriptomic and metabolomic analyses

Alginate oligosaccharides(AOS),extracted from marine brown algae,are a common functional feed additive;however,it remains unclear whether they modulate the gut microbiota and microbial metabolites.The response of Salmonella enterica serovar Typhimurium,a common poultry pathogen,to AOS fermented with chicken fecal inocula was investigated using metabolomic and transcriptomic analyses.Single-strain cultivation tests showed that AOS did not directly inhibit the growth of S.Typhimurium.However,when AOS were fermented by chicken fecal microbiota,the supernatant of fermented AOS(F-AOS)exhibited remarkable antibacterial activity against S.Typhimurium,decreasing the abundance ratio of S.Typhimurium in the fecal microbiota from 18.94 to 2.94%.Transcriptomic analyses showed that the 855 diferentially expressed genes induced by F-AOS were mainly enriched in porphyrin and chlorophyll metabolism,oxidative phosphorylation,and Salmonella infection-related pathways.RT-qPCR confrmed that F-AOS downregulated key genes involved in fagellar assembly and the type III secretory system of S.Typhimurium,indicating metabolites in F-AOS can infuence the growth and metabolism of S.Typhimurium.Metabolomic analyses showed that 205 microbial metabolites were signifcantly altered in F-AOS.Among them,the increase in indolelactic acid and 3-indolepropionic acid levels were further confrmed using HPLC.This study provides a new perspective for the application of AOS as a feed additive against pathogenic intestinal bacteria.

1例感染鼠伤寒沙门菌伴呕吐患者的护理体会

本文总结1例感染鼠伤寒沙门菌伴呕吐患者的护理经验,包括肠道传染病隔离措施、呕吐护理、心理护理、病情观察、饮食护理等护理措施,通过积极有效的护理措施预防并发症,改善患者症状,促进患者早日康复。

伤寒沙门菌外膜囊泡通过调控铁死亡抑制结直肠癌细胞的增殖及机制初探

近年来,细菌外膜囊泡(OMVs)在抗肿瘤治疗中展现出的巨大潜力引起了广泛的关注.为探究伤寒沙门菌外膜囊泡(S.Typhi-OMVs)对人结直肠癌细胞株HT-29增殖的影响及可能的作用机制,通过超速离心法提取不同细菌的OMVs,细胞增殖实验(CCK-8)检测各细菌OMVs对细胞活力的影响;转录组测序(RNA-seq)分析处理后细胞基因表达水平的变化;试剂盒检测铁死亡相关标志物的含量变化;实时荧光定量PCR(qRT-PCR)和蛋白质免疫印迹法(western blot)分别检测相关基因mRNA和蛋白质的表达变化.结果显示,在提取的6种细菌OMVs中,S.Typhi-OMVs对HT-29细胞的增殖产生了最明显的抑制作用,并且呈现出浓度和时间梯度依赖性;RNA-seq显示HT-29细胞可能发生了铁死亡;S.Typhi-OMVs作用后,细胞内发生了铁沉积,氧化产物增多,抗氧化剂减少,符合铁死亡生化特征;SAT1是S.Typhi-OMVs处理后HT-29胞内mRNA表达量变化最大的基因;p53-SAT1-ALOX15是铁死亡的信号通路之一,S.Typhi-OMVs处理后胞内p53、SAT1、ALOX15的mRNA与蛋白表达均增高.以上结果表明,S.Typhi-OMVs能够抑制结直肠癌细胞HT-29的增殖,其机制可能与通过p53-SAT1-ALOX15信号通路诱导HT-29发生铁死亡有关.

;~lasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs （shRNA） can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 （GRIM-19）. Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium （S. typhimurium） to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitroand in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

fruA基因对伤寒沙门菌侵袭力和生物膜形成的影响

为探究fruA对伤寒沙门菌生长、毒力及生物膜形成的影响,利用pBAD33制备fruA的高表达菌株(WT-pfruA)及其空载体对照菌株(WT-pBAD33).通过细菌的生长曲线测定、泳动试验、T84细胞侵袭实验以及96孔微量板结晶紫染色法分别检测fruA对伤寒沙门菌生长、毒力及生物膜形成的影响;同时应用实时荧光定量PCR (qRT-PCR)分析fruA对伤寒沙门菌毒力及生物膜相关基因的调控作用.结果显示,成功制备了伤寒沙门菌的WT-pfruA及其WT-pBAD33,发现高表达fruA后,伤寒沙门菌的生长无明显变化、运动能力增强、侵袭力上升、生物膜形成能力增加;同样地,与WT-pBAD33相比,WT-pfruA中动力、侵袭及生物膜相关基因的转录水平明显上调.综上,fruA基因可增强伤寒沙门菌的毒力及生物膜形成,并对其相关表型基因起正调控作用.

不同培养条件下伤寒沙门菌外膜囊泡对结直肠癌细胞增殖的影响

为探讨不同培养条件下伤寒沙门菌外膜囊泡(outer membrane vesicle,OMV)对结直肠癌细胞增殖的影响,本研究采用超速离心法分别提取伤寒沙门菌在正常、酸性、高渗、氧化环境LB液体培养基及LPM液体培养基中分泌的OMV,并比较其形态、粒径及所含蛋白大小。体外培养结直肠癌HT-29、SW480及CT-26细胞,采用CCK-8实验检测OMV对结直肠癌细胞增殖的影响,并用克隆形成实验进一步验证;采用流式细胞术检测细胞周期;通过统计体重及肝、肾组织苏木精-伊红染色切片评价OMV在小鼠体内的毒性。结果显示,不同培养条件下伤寒沙门菌分泌的OMV在形态、大小方面无明显差异,高渗应激环境下分泌的OMV更多。OMV所含蛋白相对分子质量在正常和酸性环境LB液体培养基中为37×10^(3)~72×10^(3),在高渗和氧化环境LB液体培养基中为25×10^(3)~72×10^(3),在LPM液体培养基中为8×10^(3)~55×10^(3)。LPM培养条件下OMV能显著抑制SW480和CT-26细胞的增殖,将SW480细胞周期阻滞于G2/M期,且对小鼠无明显肝、肾毒性。结果表明,LPM培养条件下OMV对结直肠癌细胞SW480、CT-26增殖具有直接抑制作用,有望成为结直肠癌治疗的新方案。

Identification of in vivo induced protein antigens of Salmonella enterica serovar Typhi during human infection

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.

A Salmonella enterica serovar Typhi plasmid induces rapid and massive apoptosis in infected macrophages

pRST98 is a chimeric plasmid isolated from Salmonella enterica serovar Typhi(S.typhi)that mediates the functions of drug resistance and virulence.Previously,we reported that Salmonella plasmid virulence(spv)genes were present in S.typhi.In our current study,we investigated whether plasmid pRST98 exhibits significant cytotoxicity in macrophages.pRST98 was transferred into the avirulent Salmonella enterica serovar Typhimurium(S.typhimurium)strain RIA to create the transconjugant pRST98/RIA.The standard S.typhimurium virulent strain SR-11,which carries a 100-kb virulence plasmid,was used as a positive control.The bacterial strains were incubated with a murine macrophage-like cell line(J774A.1)in vitro.Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling,and the survival of Salmonella strains in J774A.1 cells was determined.Results showed that macrophages infected with strain pRST98/RIA displayed greater levels of apoptosis than those infected with RIA and that pRST98 may increase bacterial survival in macrophages.Further studies showed that the pRST98-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pRST98 may activate caspase-9 and then caspase-3.The research data indicate that the virulence of bacteria that contain the pRST98 plasmid is enhanced;the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.

Molecular characterization and antibiotic resistance of Salmonella enterica serovar 1,4,[5],12:i:-environmental isolates from poultry farms

Salmonella enterica serovar 1,4,[5],12:i:-(S.1,4,[5],12:i:-)has been recognized as an emerging foodborne pathogen in recent years.It can cause human salmonellosis predominated by the contamination of animal-derived foods such as raw poultry and pork.This study aimed to characterize the genetic diversity,plasmid replicon types,and antibiotic resistance of 15 S.1,4,[5],12:i:-environmental isolates collected from two poultry farms using pulsed-field gel electrophoresis(PFGE),multilocus sequence typing(MLST),polymerase chain reaction-based replicon typing,and minimum inhibitory concentration approach.Ten different PFGE genotypes were detected,indicating a high diversity among these S.1,4,[5],12:i:-isolates.Three sequence types(ST19,ST1544,ST34)were identified by MLST.Among them,ST1544 was first detected in S.1,4,[5],12:i:-environmental isolates from poultry farms.All isolates were resistant to cefazolin,cefotetan,tobramycin,amikacin,and gentamicin,but susceptible to piperacillin-tazobactam,aztreonam,ceftazidime,cefepime,and ertapenem.Five incompatibility groups(Inc)of plasmids were identified,including IncFIIs(66.7%),IncHI2(20%),IncI1(6.7%),IncN(6.7%),and IncQ(6.7%).Among these isolates,80%carried at least one plasmid replicon type,and 20%carried multiple plasmid replicon types.Interestingly,the multidrug-resistant isolate 263 carried numerous resistance genes(i.e.qnrS,aac(6ʹ)-Ib-cr,bla_(TEM),bla_(CTX-M-9),bla_(OXA-1),sul1,sul2,sul3,floR,and mcr-1)and class I integronase gene intI1,which possessed both IncHI2 and IncQ plasmids,suggesting that resistance genes may be horizontally transferred by the combination of IncHI2 and IncQ plasmids.Collectively,antibiotic-resistant S.1,4,[5],12:i:-isolates were first found in poultry farm environments in China,and surveillance should be strengthened to prevent their further spread from poultry farms to foods.

Detection of Plasmid-Mediated Tigecycline Resistance Gene tet(X4) in a Salmonella enterica Serovar Llandoff Isolate

The emergence and spread of plasmid-mediated tigecycline resistance genes have attracted extensive attention worldwide.We investigated the distribution of mobile tigecycline resistance genes in Salmonella genomes generated by both our laboratory and public bacterial genomes downloaded from the NCBI GenBank.The tet(X4)-positive strains were subjected to susceptibility testing and conjugation assays.The genetic features of the tet(X4)-bearing plasmid sequence were analyzed.Here,we report the identification of the plasmid-mediated tigecycline resistance gene tet(X4)in a conjugative plasmid of the Salmonella enterica serovar Llandoff strain SH16G3606,isolated from a man in China in 2016,the first reported serovar Llandoff in China as a novel sequence type ST8300.The tet(X4)-mediated resistance phenotype was successfully transferred from the Salmonella Llandoff strain into Escherichia coli J53,resulting in a 32-fold increase in the minimal inhibitory concentration of tigecycline.The tet(X4)gene was located between two copies of ISCR2 in the plasmid pSal21GXH-tetX4.To our knowledge,this is the first report of the plasmid-mediated tigecycline resistance gene tet(X4)in a Salmonella Llandoff strain isolated from a human stool sample in China.In addition,our findings demonstrated that a total of 171 isolates are carrying tet(X)-like genes distributed in 21 countries or areas across 6 continents,posing a serious threat to humans and public health.Overall,our timely discovery of the recent emergence of the tet(X4)gene in Salmonella isolates and other Enterobacteriaceae bacteria species supports the need for rapid surveillance to prevent the tet(X)-like gene from spreading.

伤寒沙门菌基因组DNA芯片的制备与基因表达谱分析应用

伤寒沙门菌是一种具有鞭毛的革兰阴性人类肠道致病菌,也是一种重要的原核生物研究用模式菌.基因组芯片能够系统、全面且高效地观察生物的基因表达及进行基因组结构比较.利用伤寒沙门菌现有的全基因组序列,以Ty2菌株的基因组为基准,选取CT18菌株和z66阳性菌株的特异性蛋白编码基因,设计特异性引物,经PCR有效扩增出4201个基因,产物纯化后点样于多聚赖氨酸玻片制备伤寒沙门菌基因组DNA芯片,并验证了芯片样点位次与效果.通过对基因表达谱分析的各种条件进行优化,建立相应的表达谱分析方法,并用于比较伤寒沙门菌野生株在高渗、低渗条件下的基因表达差异,结果与以前的报道基本一致.结果表明,成功建立了伤寒沙门菌基因组DNA芯片及表达谱分析方法,可为有关伤寒沙门菌基因表达调控及致病性机理、进化和基因多样性等方面的深入研究提供有效的技术支持.

需氧厌氧培养方式对甲型副伤寒沙门菌检出率的影响

目的探讨需氧和厌氧血培养方式的选择对甲型副伤寒沙门菌检出率的影响。方法使用mini VI-TAL自动荧光血培养仪或BacT/Alert 3D培养仪对18684例疑似血流感染患者血液(含骨髓)标本进行需氧和(或)厌氧培养(其中仅做需氧培养935例,仅做厌氧培养16例,需氧和厌氧配对培养17733例,每瓶注入约5 ml血液)。结果18684例血液标本,共分离到甲型副伤寒沙门菌3888例(20.81%)。在17733例需氧和厌氧配对培养中检出3613例,其中仅需氧阳性406例占11.24%(406/3613),仅厌氧阳性405例占11.21%(405/3613),需氧和厌氧培养均阳性者2802例占77.55%(2802/3613),需氧和厌氧均生长的阳性报警时间分别为(22.56±13.22)h和(26.69±15.80)h,仅需氧阳性的报警时间为(32.85±23.33)h,仅厌氧阳性的报警时间为(34.46±18.44)h。结论需氧和厌氧血培养方式获得甲型副伤寒沙门菌的阳性率相同,采用需氧和厌氧瓶配对培养可提高阳性检出率,只做厌氧或需氧培养将有11.24%或11.21%阳性病例被漏检。

伤寒沙门菌phoP基因缺陷变异株的制备

目的:为深入研究伤寒沙门菌调节因子phoP的功能,制备伤寒沙门菌phoP基因缺陷变异株。方法:根据伤寒沙门菌phoP基因序列,设计PCR特异性引物,并在5′末端加接酶BglII位点,PCR扩增phoP基因上、下游片段后,用BglII消化,再定向连接成phoP基因的缺损性同源性核苷酸片段。将此片段胶回收后并导入自杀质粒pG-MB151的BamHI位点,将阳性质粒用电转化导入伤寒沙门菌野生株,进行同源重组。用PCR观察重组现象,将完全重组的菌株作为phoP基因的缺陷变异株,并通过相应的核苷酸序列分析加以确定。结果:PCR及序列分析证实,缺陷变异株的phoP基因缺失297个碱基。结论:成功构建伤寒沙门菌phoP基因缺陷变异株,为进一步研究其在伤寒沙门菌中的功能奠定了基础。

不典型山夫登堡沙门菌流行菌株的鉴定

目的对新出现的硫化氢(H2S)阴性山夫登堡沙门菌流行菌株进行鉴定。方法比较腹泻患者分离的山夫登堡沙门菌落、生化、耐药表型和沙门菌毒力岛1(SPI-1)的编码基因表型(hilA、invA),使用Riboprinter(进行核糖体指纹图谱分型,脉冲凝胶电泳(PFGE)选用XbaⅠ限制性内切酶,聚类软件选用BioNumerics软件。结果 2006年30株山夫登堡沙门菌腹泻菌株中,确认12株属于H2S和hilA、invA基因均阴性的表型不典型菌株;所有菌株除对四环素有较高耐药性(75.6%)外,对其他测试抗菌药物均较敏感;Riboprinter(核糖体分型图谱证实不典型与典型山夫登堡沙门菌在遗传学上属独立的克隆;PFGE分型将34株腹泻株分成16个带型,11株不典型菌株间存在90%的遗传学同源,优势带型为6型(6株)和4型(3株);13株典型菌株间有80%的遗传学同源,优势带型为17型(5株)、23型(5株)和11型(3株),不典型与典型菌株分别聚类成2个克隆族。结论生化、基因表型和遗传学特征不典型的山夫登堡沙门菌构成了2006年上海市山夫登堡的多点暴发病例,建议使用经过优化的常规和分子生物学方法以避免临床实验室对表型不典型沙门菌株的漏检。